Synthesis, docking and characterization of some novel 5-(S-alkyl)-1.3.4-thiadiazole-2-carboxamide derivatives as anti-inflammatory and antibacterial agents

Because of the great pharmacological and industrial significance of 1,3,4-thiadiazole and its related compounds, researchers are still very interested in them. For this reason, in this study, we looked at ways to create new hybrid compounds containing carboxamide and 1,3,4-thiadiazole moieties. The thioxoacetamide derivatives used to make these compounds were reacted with various alkylated reagents to produce multiple S-alkyl groups. Additionally, these compounds were reacted with aldehydes to form novel derivatives known as 5-(substituent)-N-phenyl-1,3,4-thiadiazole-2-carboxamide. Here, we used the agar well diffusion method to examine the antibacterial activity of all the produced compounds against a few pathogenic bacteria that were resistant to multiple drugs. Additionally, look into their capacity to lower inflammation through the use of bovine serum albumin in the protein denaturation procedure. The substances were characterized by spectral analysis (IR, 1HNMR, 13CNMR and Elemental Analysis), and efficient as antibacterial agents against all the tested bacterial strains, except for Escherichia coli. Compounds 4a and 8c showed the highest level of inhibition zone against Gram-positive bacteria (Staph. aureus, Bacillus subtilis) at concentration 0.3, 0.4 and 0.5 mg/ml compared with ciprofloxacin at the same concentrations. The results demonstrated that every compound has significant anti-inflammatory activity. At a concentration of 250 µg/ml, compounds 3a, 4c and 8c had the highest percentage inhibition of protein denaturation when (83.24%, 86.44% and 85.14%, respectively) compared to other compounds and diclofenac sodium as reference drug. Comparing compounds 4c and 8c to ciprofloxacin and diclofenac sodium, they showed powerful antibacterial and anti-inflammatory action. Furthermore, an investigation using molecular docking against DHPS from S. aureus (PDB ID: 6CLV) showed a strong connection with the intended protein and an elevated docking score, making it a prime candidate for antibiotics. Supplementary Information The online version contains supplementary material available at 10.1186/s13065-024-01237-9.


Background
Microbiological diseases are the most critical issue facing the economy and the world's health [1].It has recently become more challenging to treat bacterial infections with conventional medicines [2].Growing concern is being expressed throughout the world over the growth of bacterial resistance to well-known treatments and hospital-acquired illnesses [3].In actuality, the development of microbial resistance to commercially accessible antibacterial medications is the main cause of illness and mortality [4].Microbiological disorders that have recently caused a great deal of pain for humans include the epidemic of the plague, diphtheria, cholera, typhoid fever, a respiratory infection, and tuberculosis [5].Additionally, some recent clinical studies mention the growth in enterococci that are resistant to vancomycin, Staphylococcus epidermidis, and methicillin-resistant Staphylococcus aureus (MRSA), which are the most prevalent bacterial infections that cause death in the majority wealthy countries [6,7].As per the World Health Organisation (WHO), traditional antibiotic therapy typically fails to treat diseases caused by resistant germs, which increases the risk of mortality and lengthens suffering [8].Therefore, the development of novel antimicrobial drugs that differ from the widely used categories of antibacterial agents is still necessary [9].Moreover, one potential solution to the problem of overloaded multidrug resistance (MDR) is the development of novel drugs with distinctive mechanisms of action to prevent cross-resistance with currently available therapies [10].Because of their broad range of biological functions, heterocyclic ring structures in organic compounds continue to garner a lot of research.Numerous synthetic compounds that exhibit appealing biological effects such as antiviral [11], anticancer [12], cytotoxic [13], anticonvulsant [14], antihyperlipedemic [15], anti-inflammatory [16], analgesic [17], antidepressant [18], antioxidant [19], anti-pesticide [20], anti-COVID [21], antileishmanial [22], and antituberculosis [23] properties commonly use the scaffold 1,3,4-thiadiazole.
Many thiadiazole compounds have found extensive usage in chemotherapeutics as antimicrobial and antibacterial agents [24] that are effective against a wide range of pathogenic bacteria and resistant mycobacterium, such as compounds A and B.Moreover, mycobacterial activity has been observed to be significantly inhibited by compound B (IC 50 = 0.23 g/ml) [25].Compound C was discovered to be superior to the industry standard (pyrimethanil) when the synthetic 1,3,4-thiadiazole scaffolds were tested using the mycelial growth rate method against a few fungus strains [26].However, scaffolds D have anti-inflammatory activity and demonstrate COX-2 selectivity in the J774A.1 murine macrophage cell line [27].(Fig. 1).The impressive anti-inflammatory properties of both heterocycles and carboxamide units have been demonstrated.As a result, a lot of research has focused on creating and studying oxicam derivatives as pharmacological agents.The success of the nonsteroidal anti-inflammatory medicines (NSAIDs) piroxicam (Feldene ® ), meloxicam (Mobic ® ), and tenoxicam stimulated research in this topic (Fig. 1).Additionally, Rimonabant exerted high activity via the inhibition of COX-2 (inducible) induced at sites of inflammation [28,29].

Chemistry
As a continuation of our strategy is to determine methods to utilize these molecules as the basis for the synthesis of many different five, six, and seven-membered rings [37][38][39][40][41]. Reaction of thioxoacetamide derivatives 1a-d with carbon disulfide and potassium hydroxide in ethanol at room temperature considered an efficient method to synthesis potassium 5-(phenylcarbamoyl)-1,3,4-thiadiazole-2-thiolate derivatives (2a-d), which treated with concentrated hydrochloric acid until pH 2-3 to afford novel moiety of 1,3,4-thiadiazole derivatives 3a-d that can be used as a building block of some new 1,3,4-thiadiazole analogous (Scheme 1).The IR spectrum of compound 3a-d revealed the disappearance of NH 2 group. 1 HNMR for compound 3a showed new singles at 15.06 for NH thiadiazole group, disappeared by D 2 O, at the same time the peaks for amino group are disappeared.All the compounds show a new peak above 190 ppm in 13 C NMR which come back to C=S of the formed 1,3,4-thiadiazole rings.
Moreover, compounds 2a-c reacted with active halo compounds namely, methyl iodide ethyl iodide, 1-bromo-2-methylbutane and (bromomethyl)benzene at low temperature to give the corresponding S-alkyl derivatives with a substantial output, economical, gentle, straightforward, and environmentally friendly approach that produces suitable behaviors, see Scheme 2. Structures of the recently obtained compounds were verified based upon their IR, 1 H-NMR, 13 C-NMR, and elemental analyses.The IR spectra of compounds 4-7 exhibited the presence of broad band: at 3234-3537 cm −1 corresponding to NH groups, at 1660-1680 cm −1 corresponding to alkyl groups.The 1 H-NMR spectrum, for example, of compound 4a-c revealed the presence of a broad band at 10.60-11.03ppm characterized to NH group, a singlet signal at 2.24-2.84ppm corresponding to S-alkyl group.Where the 1 H-NMR spectrum of this compound showed a singlet signal at 11.05 ppm for NH group which disappeared by D 2 O, a multiples signals between 7.15 and 7.83 ppm for aromatic protons, singlet signal at 4.35 ppm for S-CH 2 -group, quartet signal at 4.20-4.14ppm and a triplet signal at 1.23-1.20 ppm with coupling constant equals to 7.08 Hz, which could be assigned for CH 2 CH 3 groups.The signals of 13 CNMR confirmed the expected structure by appearance of new carbonyl group at 168.08 ppm.Finally, the DEPT-135 obviously distinguished between the -CH 2 -(62.12ppm) and -CH 3 (14.56ppm) of the ethyl chain where, it showed one CH 3 with a positive phase and two CH 2 with a negative one.

Biological evaluation Antimicrobial screening
Antimicrobial activity of the tested compounds was investigated against multidrug pathogenic bacteria.The tested compounds showed potential antibacterial effect against Staph.aureus, Bacillus subtilis and K. pneumonia and no inhibitory effect against E. coli.Ciprofloxacin is used in this investigation as a control.In clinic and hospital settings, ciprofloxacin is a widely used broadspectrum antibiotic.The closest compounds to ciprofloxacin were 4c and 8c, which were more effective against Gram-positive bacteria (Staph.aureus, Bacillus subtilis) at concentration 0.3, 0.4 and 0.5 mg/ml.Furthermore, compounds 3a, 4a and 6a showed potential antibacterial effect against Staph.Aureus and Bacillus subtilis, respectively, as shown in (Table S1(supplementary file), Fig. 2).

Statistical results of antimicrobial screening
Nineteen compounds studied with different concentrations on both Gram-positive and Gram-negative bacteria, formed four subsets in accordance with the zone of inhibition values.A one-way ANOVA was conducted to compare the effect of in-vitro antibacterial activity  of compounds (Table 1).From Table 1, we have found a statistically significant result.It is observed that the in-vitro antibacterial activity of compounds 19 (Ciprofloxacin), 8c, 6a, and 4c significantly different from all other compounds.But Ciprofloxacin is used as standard.It is evident from the ANOVA that the compounds (8c, 6a, and 4c), exhibited significantly high antibacterial activity compare to the all other synthesized tested compounds and also with standard.As shown in Table 1, compound 8c exhibit significantly high antibacterial activity against S. aureus (33.26 ± 4.73) and significantly excellent antibacterial effect against Bacillus strain (36.44 ± 4.05) (Fig. 3).Moreover, compound 6a, 4c had exhibit significantly high antibacterial effect against Bacillus strain as mean = 32.66,31.54respectively.

Anti-inflammatory activity of the tested compounds
Proteins eliminate their tertiary and secondary structures when exposed to an external stressor or substance, such as a powerful base or acid, a highly concentrated inorganic salt, an organic solvent, or heating.This process is referred to as denaturation.
The expected process of denaturation is a modification in electrostatic, hydrogen, hydrophobic, and disulphide coupling.There is a dose-dependent capacity of certain anti-inflammatory medications to avoid denaturation of proteins brought about by heating [42].In this study all compounds were shown to have strong anti-inflammatory action by employing a protein denaturation inhibition technique at concentration of 50, 100, 150, 200 and 250 µg/ml in a concentration-dependent manner (Table 2).In comparison to other compounds, the compounds 3a, 4c and 8c showed the highest levels of inhibition at concentrations of 250 µg/ml with percentage inhibition 83.24%, 86.44% and 85.14%, respectively.At the same concentrations, compounds 3b and 8b exhibited significant anti-inflammatory activity with percentage inhibition 81.99% and 80.99%.These substances could therefore be a viable substitute for agents that have anti-inflammatory properties.Hence, it could be a valuable medicinal ingredient for the treatment of bacterial infections and inflammation.

Statistical results of anti-inflammatory activity
All synthesized compounds were screened for in-vitro anti-inflammatory activity by inhibition of protein denaturation method using diclofenac as a standard drug.
From Table 2, we have found a statistically significant result in all concentration (50, 100, 150, 250 µg /ml) in comparison to different test compounds.It is evident from the ANOVA that the compounds 3a, 4c, 8c, 3b

Conclusion
Synthesis, characterization, and investigation of some 1,3,4-thiadiazole derivatives which prepared from thioxoacetamide derivatives were studied, their reactions with some alkyl halides to make alkylation reaction and with some aldehydes to form novel 5-(substituent)-N-phenyl-1,3,4-thiadiazole-2-carboxamide derivatives were investigated.Finally, we studied the possibility of 1,3,4-dihydrothiadiazole derivatives as antimicrobial potential on some multidrug-resistant pathogenic bacteria.Gram-positive and Gram-negative bacteria are both targets of antimicrobial action using the agar well diffusion method then screening data is subjected to statistical analysis using one way ANOVA technique.The compounds exhibited antibacterial efficacy against all tested bacterial strains except Escherichia coli.Also, the result revealed that all compounds possessed potent significant anti-inflammatory activity.In deep study, compounds 4c and 8c possess significant antimicrobial and anti-inflammatory activity as compared to ciprofloxacin and diclofenac sodium.Additionally, a study employing molecular docking against DHPS from S. aureus (PDB ID: 6CLV) found that it is a great option for antibiotics since it is used by nearly all bacterial strains to synthesize nucleic acids.The molecular docking study exhibited positive interaction with the target protein and a high docking score especially for compounds 3a, 4c, 8d and 18a.According to the study's findings, the substances in question have strong antibacterial and anti-inflammatory properties.The overall results of this study can be considered as very promising in the perspective of new antimicrobial drugs, especially when the medical importance of tested microorganisms is considered.However, pharmacological and toxicological studies, will be necessary to confirm this hypothesis.

Chemistry
Thin layer chromatography (TLC) was employed to track all reactions utilizing percolated dishes of silica gel G/

Antimicrobial screening
According to the antibacterial activity of several compounds was screened using the agar well diffusion method [43].Ciprofloxacin was utilized to compare the results as a positive control.Dimethylsulfoxide (DMSO) solution (10% v/v) was used as a negative control.

In-vitro anti-inflammatory activity (protein denaturation) of the tested compounds
For the test compounds and the reference medication, diclofenac sodium, 0.05 mL of various concentrations (50, 100, 150, 200, and 250 µg/ml) were used, respectively.Then all tubes were combined with 0.45 ml (0.5% w/v) of BSA.The samples were heated for 3 min to maintain a temperature of 57 °C after being incubated at 37 °C for Fig. 5 2D and 3D views of the docked compounds with DHPS 20 min.Add 2.5 ml of phosphate buffer to the aforementioned solutions after cooling.At 660 nm, a UV-Visible spectrophotometer was used to detect the absorbance.Protein denaturation at 100% is represented by the control.A positive control drug called diclofenac sodium was used to compare the outcomes [44].Calculations can be made to determine the degree of protein denaturation inhibition.
A2 = Absorbance of the test sample, A1 = Absorbance of control.

Statistical analysis
Analysis was performed using Statistical Program for Social Science (SPSS) version 26 (Armonk, NY: IBM Crop).The gathering of data was recorded and evaluated on an IBM-compatible computer.One-way ANOVA was used to determine if there was any statistically significant difference.P value ≤ 0.05 was considered significant.

Molecular docking
To predict the binding style and interactions of the aforementioned drugs with dihydropteroate synthase, molecular docking experiments were carried out to better understand their efficacy (DHPS).This last one is a crucial enzyme in the prokaryotic biosynthesis of folic acid and a crucial cofactor in the pathways that almost all bacterial strains use to synthesize nucleic acids, making it a prime candidate for antibiotics [45,46].Thus, crystal structure of DHPS in complex with pterin-sulfonamide conjugates [47].PDB ID 6CLV, from S. aureus organism, was employed as a binding site in molecular docking simulations and downloaded from the RCSB protein data library.The docking results showed strong interactions with high docking scores (S) (more negative) of studied compounds to DHPS from S. aureus.The negative values of the calculated docking scores (S) for studied compounds, Table 3, demonstrates that the binding is spontaneous, and the chemicals are suitable for use as drugs [48,49].

Fig. 3
Fig. 3 Shows a comparison between test compounds and ciprofloxacin with the size of the inhibition zone of Bacillus (gram +ve) strain of bacteria Compound 3a revealed three hydrogen bonds interactions between N 7 with MET 37, S 15 with ALA 73, and O 9 with ARG 176; and 8d revealed three hydrogen bonds interactions between S 11 with ASP 42, O 21 with TYR 212, and O 22 with LYS 248; furthermore, 4c revealed three hydrogen bonds interactions between N 7 with THR 214, S % inhibition of denaturation = 100 × (1 − A2/A1) 11 with ASP 42, S 15 with ALA 41.The docking results

Table 1
In-vitro antibacterial activity of tested compounds

Table 2
Anti-denaturation activity of the tested compounds and positive control

Table 3
Docking data